1. Field of the Invention
The present invention relates to a method of preparing .alpha.-L-aspartyl-L-phenylalanine methyl ester (.alpha.-APM) which is useful as a sweetener and more specifically to an improved method of preparing .alpha.-APM by neutralizing an acid addition salt of .alpha.-APM with a base.
More precisely, it relates to a method of neutralizing an acid addition salt of .alpha.-APM in an aqueous medium, where the neutralization is effected under particular concentrations and particular temperature conditions and is followed by cooling to cause crystallization of .alpha.-APM.
2. Discussion of the Background .alpha.-APM of the present invention is a dipeptide sweetener having a sweetness of about 200 times that of sucrose (cane sugar). Because of its good quality sweetness and low calory content, it has become widely used as a diet sweetener, and the worldwide demand for it is estimated to be over 10,000 tons by 1995.
.alpha.-APM is produced industrially by several methods. In one method, an N-substituted aspartic acid anhydride and a phenylalanine methyl ester are reacted and bonded together in an organic solvent and the N-substituent is then removed from the product (U.S. Pat. No. 3,786,039). A second method of obtaining .alpha.-APM is to methyl-esterify .alpha.-L-aspartyl-L-phenylalanine in a mixed solvent comprising water, methanol and hydrochloric acid to obtain .alpha.-APM hydrochloride, and then neutralize the salt to obtain .alpha.-APM (Japanese Patent Application Laid-Open No. 53-82752). A third method of obtaining .alpha.-APM is to condense an N-substituted aspartic acid and a phenylalanine methyl ester in the presence of an enzyme and then remove the N-substituent from the product (Japanese Patent Application Laid-Open No. 55-135595).
In the first chemical synthetic method noted above, the .beta.-isomer (.beta.-L-aspartyl-L-phenylalanine methyl ester) is produced as a side product. As a means of selectively removing impurities including the .beta.-isomer, a purification method is used in which .alpha.-APM containing impurities is brought into contact with a hydrohalogenic acid and then subjected to solid-liquid separation to isolate .alpha.-APM as its hydrohalide salt.
Where industrial scale production of .alpha.-APM is required to meet the current demands, chemical methods are the major methods from the viewpoint of reducing the manufacturing cost. In this case, esterification is often effected via its hydrochloride salt as in the second method noted above, or after formation of .alpha.-APM, the hydrohalide salt is formed and purified as described above. To obtain .alpha.-APM from its hydrohalide salt, such as its hydrochloride salt, using an ordinary method, the hydrohalide salt of .alpha.-APM is dissolved or suspended in an aqueous medium and the resulting solution or suspension is neutralized by adding an aqueous solution of a base such as sodium hydroxide, sodium hydrogen carbonate or ammonia.
However, the present inventors have discovered that when a large amount, over a liter scale, of liquid must be dealt with during an industrial scale neutralization operation, such as the neutralization of an acid addition salt of .alpha.-APM, the conventional neutralization method has serious problems.
Specifically, when a base is continuously added dropwise to an aqueous solution of an acid addition salt of .alpha.-APM to reach the isoelectric point of .alpha.-APM, rapid precipitation of .alpha.-APM occurs during the course of the addition so that stirring of the system becomes impossible. In the worst case, it has been found that the stirrer completely stops. If the amount of liquid is at most 100 ml or so, as in a laboratory scale experiment, the precipitated solid phase can be easily broken with a tool, such as a spatula, whereby the fluid condition can be recovered. However, when the amount of liquid is large, i.e., about a liter or more, for example, in a bench plant or the like, or where the neutralization is carried out in a large-scale pilot plant or commercial plant, this method can not be used for solving the problem.
As a countermeasure to this problem in an industrial scale neutralization, addition of a large amount of water may be considered so as to carry out the neutralization using a diluted concentration. However, this lowers the capacity and efficiency of the device used and also lowers the yield of the product. Therefore, dilution is not a good countermeasure.
On the other hand, very slow addition of an aqueous base solution over an extremely long period of time would be effective for ensuring the fluidity of the liquid, but is ineffective from the view point of the production. Still another method is intermittently discontinuing the dropwise addition of the neutralizing agent into the reaction system having a pH range at which precipitation of .alpha.-APM starts or a pH value of 2.5 or so, to ripen the precipitated crystals (Japanese Patent Application Laid-Open No. 63-145298). This method has the serious drawback that the pH range suitable for the ripening fluctuates greatly unless the initial concentration of .alpha.-APM (or its acid addition salt) is strictly controlled to certain values. The .alpha.-APM content in the separated wet crystals (acid addition salt) always fluctuates, depending upon the delicate conditions occurring during the crystallization of the acid addition salt of .alpha.-APM. It is difficult, therefore, to keep the initial concentration of .alpha.-APM (or its acid addition salt) constant in a dissolution system controlling the amounts of liquid often used in industrial production.
In order to avoid this problem, a complicated concentration control system is necessary to effect batch-wise analysis every time and then to supply crystals or water when needed. Alternatively an expert monitor skilled in the art must be exclusively dedicated to the system to be able to batchwise determine the suitable ripening pH value each time.
Finally, even though these operational problems can be avoided by any of the above-mentioned systems, the .alpha.-APM crystals obtained still have extremely poor solid-liquid separability, which requires increased equipment costs and increased energy consumption in the filtration and drying steps used as the post-treatment steps.
A need continues to exist for a method of overcoming the above-mentioned problems in neutralizing an acid addition salt of .alpha.-APM.